Advances in health-promoting effects of natural polysaccharides: Regulation on Nrf2 antioxidant pathway

Natural polysaccharides (NPs) possess numerous health-promoting effects, such as liver protection, kidney protection, lung protection, neuroprotection, cardioprotection, gastrointestinal protection, anti-oxidation, anti-diabetic, and anti-aging. Nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway is an important endogenous antioxidant pathway, which plays crucial roles in maintaining human health as its protection against oxidative stress. Accumulating evidence suggested that Nrf2 antioxidant pathway might be one of key regulatory targets for the health-promoting effects of NPs. However, the information concerning regulation of NPs on Nrf2 antioxidant pathway is scattered, and NPs show different regulatory behaviors in their different health-promoting processes. Therefore, in this article, structural features of NPs having regulation on Nrf2 antioxidant pathway are overviewed. Moreover, regulatory effects of NPs on this pathway for health-promoting effects are summarized. Furthermore, structure-activity relationship of NPs for health-promoting effects by regulating the pathway is preliminarily discussed. Otherwise, the prospects on future work for regulation of NPs on this pathway are proposed. This review is beneficial to well-understanding of underlying mechanisms for health-promoting effects of NPs from the view angle of Nrf2 antioxidant pathway, and provides a theoretical basis for the development and utilization of NPs in promoting human health.

Frontiers in Nutrition 02 frontiersin.org Molecular mechanism of Nrf2 signaling pathway regulating oxidative stress (7,8 Nrf2 translocates quickly into nucleus and forms a necessary region for the dimer by binds to small musculoaponeurotic fibrosarcoma oncogene homolog (sMaf) protein. Subsequently, this region binds to antioxidant response elements (ARE) and activates the expressions of target genes, thereby regulates the transcriptional activities of phase II metabolic enzymes, antioxidant enzymes or drug transporters, for restoring intracellular redox homeostasis. Recently, a variety of natural products, such as polyphenols, flavonoids and polysaccharides, have been considered as modulators of Nrf2 antioxidant pathway (9,10). Polysaccharides, a kind of biological macromolecules, are widely distributed in natural sources such as plants, algae and animals (11). Polysaccharides have attracted increasing attention owing to their diverse health-promoting effects, non-toxicity, extensive accessibility and renewability (12). Polysaccharides from natural resources (NPs) have been reported to play key roles in regulating excessive oxidative stress (13). In the past few decades, regulations of NPs on Nrf2 antioxidant pathway have been extensively studied in their health-promoting effects, such as liver protection (14), antioxidant (15), gastrointestinal protection (16), anti-diabetic (17), anti-aging (18), cardioprotection (19), lung protection (20), kidney protection (21), neuroprotection (22), anti-inflammation (23), immunomodulation (24), anti-depression (25), anti-cancer (26), improving reproductive function (27), anti-radiation (28), and anti-atherosclerosis (29). However, the information concerning regulation of NPs on Nrf2 antioxidant pathway is scattered, and NPs show different regulatory behaviors in their different health-promoting processes. Therefore, it is necessary to draw a summary on the recent developments on health-promoting effects of NPs from the viewing angle of regulation on Nrf2 antioxidant pathway.
In this review, structural features of NPs, having regulation on Nrf2 antioxidant pathway, from herbs, woody plants, algae, fungi, animals and bacteria, are overviewed. Moreover, regulatory effects of these NPs on the pathway for health-promoting effects in vitro and in vivo are systematically summarized. Furthermore, influences of structural characteristics like molecular weight (M w ), functional group, monosaccharide composition and side chains on the regulatory effects of NPs on Nrf2 antioxidant pathway are preliminarily discussed. Otherwise, the prospects on future work for regulation of NPs on Nrf2 antioxidant pathway are proposed.

Structural features of NPs from animals and bacteria
In terms of NPs having regulation on Nrf2 antioxidant pathway from animals, structural features of polysaccharides from Holothuria leucospilota (114), Acaudina leucoprocta (115), and Ostrea talienwhanensis Crosse (42,43)   However, the obtained purified NPs usually exhibited different structural features, owing to different methods and protocols      used in above isolation and purification processes. Acidic polysaccharides (CPP-1 and CPSP-1; CTP-1 and CTSP-1) purified respectively from roots (55) and stems (66) of Codonopsis pilosula and Codonopsis tangshen had different M w , monosaccharide composition, glycosidic bond types, backbone and side chains. Two purified fractions (TTP-1 and TVP) acquired from tubers (71) and vines (86) of Tetrastigma hemsleyanum revealed differences in M w and monosaccharide composition. A low-fucose-content polysaccharide (LFC) (53) and a high-fucose-content one (HFC) (37, 38) were purified from the glucose mineral salts medium (GMSM) and one in GMSM-supplemented jute culture of Bacillus megaterium, and they displayed different M w , monosaccharide composition, glycosidic bond types, backbone and side chains. Two polysaccharides (PNP80b-2 and PNP40c-1) were purified from water extracts of Pinus koraiensis pine nut by ethanol (80 and 40%, respectively) precipitation and same column chromatography procedures, and they were different in M w , monosaccharide composition and glycosidic bond types (87)(88)(89)(90). Two purified fractions (EPP80 and EPPS-3) from Echinacea purpurea were obtained by ultrasonic extraction and stepwise ethanol precipitation (36), and water extraction and column chromatography (116), respectively. EPP80 and EPPS-3 exhibited different M w and monosaccharide composition. Two fractions (DRP1 and DRP2) from Dandelion root polysaccharides were obtained by column chromatography with water and 0.1 M NaCl elution, respectively, and they showed differences in M w , monosaccharide composition, glycosidic bond types and backbone (57). Five purified fractions (PS-1, PS-2, PS-3, PS-4, and PS-5) were gained from Athyrium multidentatum subsequently eluted with 0, 0.1, 0.2, 0.3, and 0.4 M NaCl solutions, and they possessed different M w and monosaccharide composition ratios (85). Two purified polysaccharides (CPP0.05 and CPP0.1) were obtained by eluting with 0.05 M and 0.1 M NaCl from Cyclocarya paliurus, and they behaved differences in M w , monosaccharide composition, glycosidic bond types, backbone and side chains (72,95,96).

Regulation of NPs from algae
The regulations of NPs on Nrf2 antioxidant pathway from algae in cell and animal experiments are revealed in Table 3.

Regulation of NPs from fungi
The regulations of NPs on Nrf2 antioxidant pathway from fungi in cell experiments and animal experiments are illustrated in Table 4.
Polysaccharides from Bacillus megaterium could regulate Nrf2 antioxidant pathway for lung protection (38) and anti-cancer (53), as listed in Table 5. Cell experiments have demonstrated that this polysaccharide exerted lung protection on H 2 O 2 -induced WI38 cells by enhancing protein expressions of cytosol Keap1 and cytosol Nrf2, and suppressing protein expressions of nuclear Keap1 and Nrf2 as well as nuclear translocation of Nrf2 (38). Meanwhile, the polysaccharide exhibited anti-cancer effect on A549 cells through increasing protein expressions of cytosol Keap1 and Nrf2, and decreasing protein expressions of nuclear Keap1 and Nrf2 (53).
With above analyses, regulations of NPs on Nrf2 antioxidant pathway in health-promoting effects in vitro and in vivo can be summarized in Figures 2, 3, respectively.
Structure-activity relationship of NPs for health-promoting effects by regulating Nrf2 antioxidant pathway Structure-activity relationship of NPs for health-promoting effects by regulating Nrf2 antioxidant pathway is unclear. However, the influences of M w , functional group, monosaccharide composition and side chains on the efficacies of NPs in regulating Nrf2 antioxidant pathway could be preliminarily discussed.

Influence of M w
There might be two different standpoints concerning the influence of M w on the regulation of NPs to Nrf2 antioxidant pathway. One standpoint is that polysaccharide with higher M w generated stronger regulation on Nrf2 antioxidant pathway in vitro and in vivo. Polysaccharide (AZP-1a) with higher M w (34.1 kDa) from Anoectochilus zhejiangensis exhibited better protection on CCl 4 -treated HepG2 cells than that (AZP-1d) with lower M w (4.     (208). Another standpoint is that polysaccharide with lower M w caused stronger regulation on Nrf2 antioxidant pathway in vitro and in vivo. Polysaccharide (TOP-2) with smaller M w (<1 kDa) from Taraxacum officinale elevated more protein expressions of Nrf2 and HO-1 than that (TOP-1) with larger M w (1-9.3 kDa) in LPS-induced RAW264.7 cells, although TOP-2 and TOP-1 had no significance in protecting RAW264.7 cells (132). Polysaccharide (DRP1) with lower M w (5.695 kDa) from Dandelion root reflected better hepatoprotection on CCl 4 -induced liver injury in mice than that (DRP2) with higher M w (8.882 kDa). Meanwhile, DRP1 increased relatively more mRNA expressions of Nrf2 and NQO1 while decreased more mRNA expression of Keap1 in the liver than DRP2 (57). Polysaccharide (FWBP, 21.19 kDa) from fermented wheat bran has been shown to be more effectiveness in positively regulating gut antioxidant-associated gene expression and gut microbiota in zebrafish than that (WBP, 52.03 kDa) from wheat bran. At the same time, FWBP produced more mRNA expressions of CAT, GST, and Nrf2 along with less GPX-3 mRNA expression than than WBP in zebrafish (162). Two different polysaccharides (CPSP-1, 13.1 kDa; CTSP-1, 23.0 kDa) have been obtained from stems of Codonopsis pilosula and Codonopsis tangshen, respectively (66). CPSP-1 showed higher protective effect on H 2 O 2 -induced IPEC-J2 cells and had a better promotion on GPXs and SOD1 expressions than CTSP-1. Meanwhile, a polysaccharide (CPP-1) with M w of 21.0 kDa from Codonopsis pilosula roots showed stronger protection on H 2 O 2 -induced IPEC-J2 cells and regulation on Nrf2 antioxidant pathway than that (CTP-1) with M w of 29.5 kDa from Codonopsis tangshen roots (55).
However, polysaccharide with moderate M w might be more beneficial to regulate Nrf2 antioxidant pathway. For example, Han et al. (60) have investigated the repair effects of three Astragalus polysaccharides (APS0, APS1, and APS2) with different M w (11.03, 4.72, and 2.61 KDa) against oxalate-induced HK-2 cells. The findings displayed that APS1 with the moderate M w provided the strongest repair effect and increased the most protein expressions of Keap1, Nrf2, SOD1, and CAT.

Influence of functional group
Selenization, sulfuration, and acetylation modifications could improve the regulation of NPs on Nrf2 antioxidant pathway, owing to new functional groups have been brought in. Selenizing Codonopsis pilosula polysaccharides (sCPPS 5 ) caused significantly stronger protective effect on H 2 O 2 -induced RAW264.7 cells and more increases in protein expressions of Nrf2, HO-1, NQO1, GCLM, and GCLC and declination in Keap1 protein expression than unmodified polysaccharide (CPPS) (131). Selenizing Astragalus polysaccharides (sAPS) exhibited markedly higher protection against CCl 4 -induced liver injury in rats and up-regulated more mRNA expression levels of GPX1, SOD1 and Nrf2 in the liver than the native one (APS) (150). On the other hand, sulfated Cyclocarya paliurus polysaccharide (S-CPP 0.05 ) showed stronger antioxidant activity to H 2 O 2 -induced DCs and generated more increment in Nrf2 protein expression and reduction in Keap1 protein expression in DCs, as compared with the native one (CPP 0.05 ) (96). At the dosages of 100 and 200 mg/kg, sulfated Codonopsis polysaccharide (SCP) produced better hepatoprotective effect on liver in ethanol-induced mice and more decreases in mRNA expressions of Nrf2 and Keap1 than the native one (CP) in the liver (164). Otherwise, acetylated Cyclocarya paliurus polysaccharide (Ac-CPP 0.1 ) generated higher cytoprotection on H 2 O 2 -induced DCs and improved more mRNA expressions of SOD1, GPX1, CAT, HO-1, and NQO1 than the native one (CPP 0.1 ) (72). Acetylated Stropharia rugoso-annulata polysaccharides (ASRP) exhibited better action in alleviating non-alcoholic fatty liver in HFD-induced mice and caused more HO-1 protein expression and less Keap1 protein expression in liver tissues (209).

Influence of monosaccharide composition
Natural polysaccharides with higher GalA or GlcA may cause better regulation effect on Nrf2 antioxidant pathway. Two polysaccharides (CPSP-1 and CTSP-1) gained from stems of Codonopsis pilosula and Codonopsis tangshen were determined to contain GalA of 70.1 and 61.3%, respectively. The former was proven to have better protective action on H 2 O 2 -induced IPEC-J2 cells and regulation effect on Nrf2 antioxidant pathway (66). Five fractions (PS-1, PS-2, PS-3, PS-4, and PS-4) from Athyrium multidentatum were characterized to contain GlcA content with an order as PS-1 < PS-5 < PS-4 < PS-2 < PS-3 (85). PS-1 showed the lowest cytoprotection on H 2 O 2 -induced HUVECs cells and regulation on mRNA expressions of Nrf2 and HO-1. Two purified polysaccharides (RGP-1-A and RGP-2-A) obtained from Rehmannia glutinosa were determined to have GalA contents of 19.02 and 1.1%. RGP-1-A showed significantly better cytoprotection on H 2 O 2 -induced IPEC-1 cells and caused observably more increments in mRNA expressions of Nrf2, HO-1 and NQO1 and reduction in Keap1 mRNA expression (208).
On the other hand, higher contents of Ara, Gal, and Rha may have greater regulation effect on Nrf2 antioxidant pathway. The polysaccharides (CPP-1 and CTP-1) from roots of Codonopsis pilosula and Codonopsis tangshen contained Ara+Gal+Rha contents of 41.1 and 39%, respectively. CPP-1 revealed relatively protection on H 2 O 2 -induced IPEC-J2 cells and greater regulation on Nrf2 antioxidant pathway (55). Meanwhile, the above-mentioned PS-1 with smallest Ara+Gal+Rha contents showed the lowest cytoprotection on H 2 O 2 -induced HUVECs cells and regulation on mRNA expressions of Nrf2 and HO-1, as compared with PS-2, PS-3, PS-4, and PS-5 (85).

Influence of side chains
Shorter AG side chains of NPs can be more effective in promoting Nrf2 antioxidant pathway. A polysaccharide (CPSP-1) with AG-II chains acquired from Codonopsis pilosula stems showed stronger protective effect on H 2 O 2 -induced IPEC-J2 cells and promotion on Nrf2 antioxidant pathway than that (CTSP-1) with AG-I and AG-II chains from Codonopsis tangshen stems (66). Moreover, CPP-1 with shorter AG-I chains from Codonopsis pilosula roots revealed better protection on H 2 O 2 -induced IPEC-J2 cells and regulation on Nrf2 antioxidant pathway than CTP-1 with longer AG-I chains from Codonopsis tangshen roots (55).

Conclusions and prospects
This review summarizes that NPs from natural sources can regulate Nrf2 antioxidant pathway to exert a wide spectrum of health-promoting effects in vitro and in vivo, such as liver protection, kidney protection, lung protection, neuroprotection, cardioprotection, gastrointestinal protection, anti-oxidation, anti-diabetic, anti-aging, anti-inflammation, anti-radiation, antidepression, anti-cancer, anti-atherosclerosis, immunomodulation, and improving reproductive function. Moreover, some factors like Keap1, Nrf2, HO-1, NQO1, GCLC, GCLM, γ-GCL, γ-GCS, γ-GCSc, Mn-SOD, SODs, GPXs, CAT, GST, Gstm1, Gstt1, and PGC-1α in Nrf2 antioxidant pathway are modulated in the frequently seen in vitro health-promoting effects (liver protection, kidney protection, lung protection, cardioprotection, gastrointestinal protection, anti-oxidation, anti-diabetic and antiaging) of NPs (Figure 2). Meanwhile, Keap1, Nrf2, HO-1, NQO1, GCLC, GCLM, γ-GCS, Cu/Zn-SOD, Mn-SOD, SODs, GPXs, GR, CAT, GSTs, NOX2, NOX4, TrxR1, Slc7a11, G6pd2, Prdx1, PGC-1α, MKP1, and p22/47/67phox are regulated in these in vivo healthpromoting effects (Figure 3). On the other hand, NPs having regulation on Nrf2 antioxidant pathway can be widely acquired by water extraction and column chromatography methods. Although many studies have disclosed the regulation of NPs on Nrf2 antioxidant pathway, there are still some problems should be explored in future: (i) compared with NPs from herbs and woody plants, less researches have been conducted to the regulative effects of NPs from algae, fungi, animals, and bacteria on Nrf2 antioxidant pathway; (ii) existing evidences are inadequate to establish structure-activity relationship for regulation of NPs on Nrf2 antioxidant pathway in their health-promoting effects; (iii) clinical research on the regulation of NPs on Nrf2 antioxidant pathway is scarce, and regulation of NPs on Nrf2 antioxidant pathway is rarely reported in some health-promoting effects; (iv) Nrf2 antioxidant pathway is activated by NPs in most cases, whilst it is inhibited by NPs in several health-promoting effects like anti-cancer. However, there is few information concerning the classification of NPs as activators and inhibitors; (v) as shown in Tables 1-5, regulation of NPs on Nrf2 antioxidant pathway has been determined by WB, RT-PCR, RT-qPCR, IHC, IF, ChIP, EMSA, and ELISA as well as assay kits. However, Nrf2 antioxidant pathway is a complex network and it has some relations with other pathways. Thus, proteomics, transcriptomics and other methods can be used to explore the regulation of NPs on Nrf2 antioxidant pathway; (vi) there are many genes like PI3K, JNK, ERK, and AKT can regulate Nrf2 antioxidant pathway (10), the effects of NPs on these genes should also be explored; (vii) which procedure is more suitable for preparing NPs with regulation on Nrf2 antioxidant pathway, and which structure has the stronger regulation, cannot be concluded.